1. 2×Universal Blue SYBR Green qPCR Master Mix With 40×Yellow Template Dilution Buffer, 1ml $99,

Product Information

Description/Introduction

This product is a 2x premix solution for qPCR using SYBR Green I chimeric fluorescence method. The core component, Taq DNA Polymerase, is a heat-activated DNA Polymerase blocked by antibody method, which can effectively inhibit nonspecific amplification under low temperature conditions. Meanwhile, combined with the reaction Buffer optimized for qPCR, Taq DNA Polymerase is very suitable for high specificity and sensitivity qPCR reaction. A good standard curve can be obtained in a wide quantitative area, which is accurate, reproducible and reliable for the quantitative analysis of target genes. At the same time, this product contains a special ROX Passive Reference Dye which is suitable for use in all qPCR instruments, and there is no need to adjust the ROX concentration on different instruments. Blue dye is added in the premix, which has the tracer effect of adding samples, and yellow template diluent is provided at the same time. When the template is diluted with yellow template diluent and added into the blue reaction premix, the color changes from blue to green, which can play a tracer role in the preparation of reaction system process and prevent leakage or misaddition. The spectra of the blue and yellow dyes do not overlap with the qPCR dyes and do not affect the reaction results.

Storage and Handling Conditions

Wet ice bag transportation; Stored at -20℃ temperature, valid for 12 months.

Component

Assay Protocol / Procedures

1. (Optional) template dilution

This kit provides a 40x YELLOW Template Dilution Buffer, a 40-fold diluted Yellow Template Dilution that can be used to accurately determine whether the Template has been added to the qPCR reaction fluid by changing the color of the liquid. Taking the 20μL qPCR reaction system as an example, according to the dilution template amount added into the 20μL qPCR reaction solution, the corresponding dilution method of the original template can be referred to the following table (taking the original template diluted to 100μL as an example):

Example: 2μL diluted template should be added to 20μL qPCR reaction system. Taking the 100μL Template Dilution system as an example, when the original Template volume is 20μL, 25μL 40x Yellow Template Dilution Buffer is added, and then Nuclease-free Water 55μL is added to the total volume of 100μL. The 40x YELLOW TEMPLATE DILUTION BUFFER is now 10x. Add 2μL of the diluted Template into a 20μL Dilution Dilution Buffer, and the final 40× YELLOW Template Dilution Buffer is 1x. In conclusion, the Yellow Template Dilution Buffer should be 1x in the final qPCR system.

Note: If the template does not need to be diluted, or if the template diluent of G3362-2 is not used, you can ignore this step.

2. Recommend the qPCR reaction system:

a. Usually, a good amplification effect can be obtained with the final concentration of 0.2μM. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1.0μM.

b. The amount of template addition varies with the copy number of the target gene in the template solution, and the appropriate amount of template addition is discussed by gradient dilution. The best addition amount of template DNA in the 20μL reaction system was less than 100 ng. When the cDNA (RT reaction solution) of RT-PCR reaction was used as template, the addition amount should not exceed 10% of the total volume of PCR reaction solution.

3. PCR reaction procedure (can be adjusted according to the instruments) :

a: If amplification specificity needs to be improved, two-step procedure or annealing temperature can be used; To improve the amplification efficiency, a three-step procedure or extension time can be used.

Note:

1. Please wear disposable gloves during operation to avoid RNase contamination.

2. The reverse transcription products can be stored at -20℃ for a short time. If long-term storage is needed, it is recommended to store them at -80℃ after packing to avoid repeated freeze-thaw cycles.

3. If the template is of eukaryotic origin, it is recommended to select Oligo (dT)18 Primer and pair it with the 3′ Poly A tail of eukaryotic mRNA to obtain the highest yield of full-length cDNA.

4. For reverse transcription of prokaryotic RNA, Random Hexamer Primer or Gene Specific Primer should be used.

5. Random Hexamer Primer has wide applicability and is suitable for mRNA, rRNA, tRNA, small RNA and lncRNA templates.

6. If reverse transcription is followed by qPCR assay, Oligo (dT)18 Primer and Random Hexamer Primer can be mixed to make cDNA synthesis efficiency in all regions of mRNA the same, which helps to improve the authenticity and repeatability of quantitative results.

7. If the subsequent qPCR primers are designed across exons, the genome removal step can be omitted.

Compatibility:

ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900 HT Fast, StepOne™, StepOne Plus™, 7500/7500 Fast, ViiA 7™, QuantStudio™ series, PikoRealTM Cycler;

Stratagene: Mx3000P®, 3005P™, 4000™;

Bio-Rad: CFX96™, CFX384™, iCycler iQ™, iQ5™, MyiQ™, MiniOpticon™, Opticon®, Opticon 2, Chromo4™;

Eppendorf: Realplex 2s, Mastercycler® ep, realplex;

IIIumina: Eco QPCR;

Cepheid: SmartCycler®;

Qiagen Corbett: Rotor-Gene® series;

Roche: LightCycler™ series;

Takara: Thermal Cycler Dice series;

Analytikjena: qTOWER series;

qTOWER: LineGene series.

Primer design principles:

1. The length of amplification product is recommended to be between 80-300 bp;

2. Primer length: 18-25 bp;

3. The content of base G+C in primers should be between 40%-60%;

4. The Tm value difference between forward primers and reverse primers is less than 2℃, and the Tm value between 58-62℃ is the best;

5. Randomness of base distribution;

6. Primers had better not contain self-complementary sequences, otherwise they will form a secondary hairpin structure;

7. There should be no more than 4 complementary or homologous bases between two primers, otherwise primer dimer will be formed, especially complementary overlap at the 3′ end;

8. The 3′ terminal base of the primer is suggested to be G or C;

9. No other non-specific products were found in NCBI comparison results.

Common problems and solutions:

For Research Use Only!

Ver. No.: V1.0-202111